Experimental activation of telomerase was recently shown to extend the in vitro replicative life-span of certain primary human cells, demonstrating that replicative senescence is controlled by telomere length. The experiments proposed here aim to elucidate the molecular mechanism by which the shortening of human telomeres induces senescence in vitro. We have identified four protein components of the human telomeric complex, two duplex telomeric binding proteins (TRF1 and TRF2), tankyrin, which interacts with TRF1, and HC1, a TRF2-interacting protein. Our preliminary evidence suggests that one of these factors, TRF2, may be involved in the regulation of telomere-induced senescence. We will use these four telomeric proteins in molecular genetic experiments to test three possible models for how telomere shortening might signal to the senescence pathway. The models are: (1) Telomere shortening creates a pool of free telomeric proteins that signal senescence. (2) Telomeres contain an inducer of senescence that is repressed by a second telomeric protein. As a consequence of telomeric shortening the repressor is lost, resulting in activation of a senescence program by the telomeric inducer. (3) Telomere shortening leads to the unmasking of the chromosome ends, activating a DNA damage checkpoint that induces senescence. Each of these models will be tested through the overexpression and inhibition of telomeric proteins using viral expression systems in primary human cells (AIMs 1-4). In AIMs 5 and 6, the resulting phenotypes will be characterized in detail. Specifically, if a premature senescence-like phenotype is observed in response to overexpression or inhibition of telomeric proteins, we will use molecular, cytological, and cell biological techniques to compare this phenotype to scheduled, natural senescence in the same cell strain. In addition we will use microarray techniques in order to query the expression patterns of over 10,000 genes in normal and premature telomere-induced senescence. These experiments are expected to reveal the molecular pathway that allows cells to respond to the telomeric clock. Definition of this, as yet unknown, signaling pathway should reveal the nature of senescence and facilitate further analysis of its relevance to human aging.